Overproduction of transforming growth factor (TGF).beta. clearly underlies tissue fibrosis caused by excess deposition of extracellular matrix resulting in disease. TGF.beta.'s fibrogenic action results from simultaneous stimulation of matrix protein synthesis, inhibition of matrix degradation and enhanced integrin expression that facilitates extracellular matrix (ECM) assembly. Overproduction of TGF.beta. has been demonstrated in glomerulonephritis, diabetic nephropathy and hypertensive glomerular injury. Suppression of the production of ECM and prevention of accumulation of mesangial matrix in glomeruli of glomerulonephritic rats has been demonstrated by intravenous administration of neutralizing antibodies specific for TGF.beta. (Border et al., Nature 346:371-374 (1990)) or administration of purified decorin (Border et al., Nature 360:361-364 (1992)) and by introduction of nucleic acid encoding decorin, a TGF.beta.-inhibitory agent, into a rat acute mesangial model of glomerulonephritis (Isaka et al., Nature Med. 2:418-423 (1996)).
Renin is an aspartyl proteinase synthesized by juxtaglomerular kidney cells and mesangial cells in humans and rats. (Chansel et al., Am. J. Physiol. 252:F32-F38 (1987) and Dzau and Kreisberg, J. Cardiovasc. Pharmacol. 8(Suppl 10):S6-S10 (1986)). Renin plays a key role in the regulation of blood pressure and salt balance. Its major source in humans is the kidney where it is initially produced as preprorenin. Signal peptide processing and glycosylation are followed by secretion of prorenin and its enzymatically active form, mature renin. The active enzyme triggers a proteolytic cascade by cleaving angiotensinogen to generate angiotensin I, which is in turn converted to the vasoactive hormone angiotensin II by angiotensin converting enzyme ("ACE").
The sequence of the human renin gene is known (GenBank entry M26901). Recombinant human renin has been synthesized and expressed in various expression systems (Sielecki et al., Science 243:1346-1351 (1988), Mathews et al., Protein Expression and Purification 7:81-91 (1996)). Inhibitors of renin are known (Rahuel et al., J. Struct. Biol. 107:227-236 (1991); Badasso et al., J. Mol. Biol. 223:447-453 (1992); and Dhanaraj et al., Nature 357:466-472 (1992)) including an orally active renin inhibitor in primates, Ro 42-5892 (Fischli et al., Hypertension 18:22-31 (1991)). Renin-binding proteins and a cell surface renin receptor on human mesangial cells have been identified (Campbell and Valentijn, J. Hypertens. 12:879-890 (1994), Nguyen et al., Kidney Internat. 50:1897-1903 (1996) and Sealey et al., Amer. J. Hyper. 9:491-502 (1996)).
The renin-angiotensin system (RAS) is a prototypical systemic endocrine network whose actions in the kidney and adrenal glands regulate blood pressure, intravascular volume and electrolyte balance. In contrast, TGF.beta. is considered to be a typical cytokine, a peptide signaling molecule whose multiple actions on cells are mediated in a local or paracrine manner. Recent data however, indicate that there is an intact RAS in many tissues whose actions are entirely paracrine and TGF.beta. has wide-ranging systemic (endocrine) effects. Moreover, RAS and TGF.beta. act at various points to regulate the actions of one another.
In a systemic response to an injury such as a wound, the RAS rapidly generates AII that acts by vasoconstriction to maintain blood pressure and later stimulates the secretion of aldosterone, resulting in an increase in intravascular volume. In the wound, TGF.beta. is rapidly released by degranulating platelets and causes a number of effects including: 1) autoinduction of the production of TGF.beta. by local cells to amplify biological effects; 2) chemoattraction of monocyte/macrophages that debride and sterilize the wound and fibroblasts that begin synthesis of ECM; 3) causing deposition of new ECM by simultaneously stimulating the synthesis of new ECM, inhibiting the proteases that degrade matrix and modulating the numbers of integrin receptors to facilitate cell adhesion to the newly assembled matrix; 4) suppressing the proinflammatory effects of interleukin-1 and tumor necrosis factor; 5) regulating the action of platelet derived growth factor and fibroblast growth factor so that cell proliferation and angiogenesis are coordinated with matrix deposition; and 6) terminating the process when repair is complete and the wound is closed (Border and Noble, Scientific Amer. Sci. & Med. 2:68-77 (1995)).
Interactions between RAS and TGF.beta. occur at both the systemic and molecular level. It has been shown that TGF.beta.'s action in causing ECM deposition in a healing wound is the same action that makes TGF.beta. a powerful fibrogenic cytokine. (Border and Noble, New Engl. J. Med. 331:1286-1292 (1994); and Border and Ruoslahti, J. Clin. Invest. 90:107(1992)). Indeed, it is the failure to terminate the production of TGF.beta. that distinguishes normal tissue repair from fibrotic disease. RAS and TGF.beta. co-regulate each other's expression. Thus, both systems may remain active long after an emergency response has been terminated, which can lead to progressive fibrosis. The kidney is particularly susceptible to overexpression of TGF.beta.. The interrelationship of RAS and TGF.beta. may explain the susceptibility of the kidney to TGF.beta. overexpression and why pharmacologic suppression of RAS or inhibition of TGF.beta. are both therapeutic in fibrotic diseases of the kidney. (Noble and Border, Sem. Nephrol., supra and Border and Noble, Kidney Int. 51:1388-1396 (1997)).
Activation of RAS and generation of angiotensin II (AII) are known to play a role in the pathogenesis of hypertension and renal and cardiac fibrosis. TGF.beta. has been shown to be a powerful fibrogenic cytokine, acting simultaneously to stimulate the synthesis of ECM, inhibit the action of proteases that degrade ECM and increasing the expression of cell surface integrins that interact with matrix components. Through these effects, TGF.beta. rapidly causes the deposition of excess ECM. AII infusion strongly stimulates the production and activation of TGF.beta. in the kidney. (Kagami et al., J. Clin. Invest. 93:2431-2437 (1994)). Angiotensin II also upregulates TGF.beta. production and increases activation when added to cultured vascular smooth muscle cells (Gibbons et al, J. Clin. Invest. 90:456-461 (1992)) and this increase is independent of pressure (Kagami et al., supra). Blockade of AII reduces TGF.beta. overexpression in kidney and heart, and it is thought that TGF.beta. mediates renal and cardiacfibrosis associated with activation of RAS (Noble and Border, Sem. Nephrol. 17(5):455-466 (1997)). Blockade of AII using inhibitors of ACE slow the progression of renal fibrotic disease (see, e.g., Anderson et al., J. Clin. Invest. 76:612-619 (1985) and Noble and Border, Sem. Nephrol. 17(5):455-466 (1997)). What is not clear is whether angiotensin blockade reduces fibrosis solely through controlling glomerular hypertension and thereby glomerular injury, or whether pressure-independent as well as pressure-dependent mechanisms are operating. While ACE inhibitors have been shown to slow the progress of fibrotic diseases, they do not halt disease and TGF.beta. levels remain somewhat elevated.
Thus, RAS and TGF.beta. can be viewed as powerful effector molecules that interact to preserve systemic and tissue homeostasis. The response to an emergency is that RAS and TGF.beta. become activated. Continued activation may result in chronic hypertension and progressive tissue fibrosis leading to organ failure. Because of the interplay between the RAS and TGF.beta., and the effects of this interplay on tissue homeostasis, blockade of the RAS may be suboptimal to prevent or treat progressive fibrotic diseases such as diabetic nephropathy.
Components of the renin-angiotensin system act to further stimulate production of TGF.beta. and plasminogen activator inhibitor leading to rapid ECM accumulation. The protective effect of inhibition of the renin-angiotensin system in experimental and human kidney diseases correlates with the suppression of TGF.beta. production.
The renin molecule has been shown to enzymatically cleave angiotensinogen into Angiotensin I. The angiotensin I is then converted by Angiotensin Converting Enzyme ("ACE") to Angiotensin II which acts as an active metabolite and induces TGF.beta. production. Angiotensin II is an important modulator of systemic blood pressure. It has been thought that if you decrease hypertension by blocking AII's vasoconstrictor effects fibrotic disease is reduced.
In the glomerular endothelium, activation of RAS and TGF.beta. have been shown to play a role in the pathogenesis of glomerulonephritis and hypertensive injury. Volume (water) depletion and restriction of potassium have been shown to stimulate both production of renin and TGF.beta. in the juxtaglomerular apparatus (JGA) of the kidney (Horikoshi et al., J. Clin. Invest. 88:2117-2122 (1992) and Ray et al., Kidney Int. 44:1006-1013 (1993)). ACE inhibitor has also been shown to increase the production of renin and TGF.beta., suggesting that AII is not inducing TGF.beta. but that production of renin and TGF.beta. are co-regulated. TGF.beta. has been shown to stimulate the release of renin from kidney cortical slices and cultured JG cells (Antonipillai et al., Am. J. Physiol. 265:F537-F541 (1993); Ray et al., Contrib. Nephrol. 118:238-248 (1996) and Veniant et al., J. Clin. Invest. 98:1996-19970 (1996)). Other interactions between RAS and TGF.beta. include that AII induces the production of TGF.beta. in cultured cells and in vivo (Kagami et al., supra). It is thus likely that the fibrogenic effects that have been attributed to AII are actually mediated by TGF.beta..
Another interplay between RAS and TGF.beta. is with the production of aldosterone. Aldosterone overproduction has been linked to hypertension and glomerulosclerosis. AII stimulates the production and release of aldosterone from the adrenal gland. In contrast, TGF.beta. suppresses aldosterone production and blocks the ability of AII to stimulate aldosterone by reducing the number of AII receptors expressed in the adrenal (Gupta et al., Endocrinol. 131:631-636 (1992)), and blocks the effects of aldosterone on sodium reabsorption in cultured collecting renal duct cells (Husted et al., Am. J. Physiol. Renal, Fluid Electrolyte Physiol. 267:F767-F775 (1994)). Aldosterone may have fibrogenic effects independent of AII, and may upregulate TGF.beta. expression. The mechanism of aldosterone's pathological effects is unknown but might be due to stimulation of TGF.beta. production in the kidney (Greene et al., J. Clin. Invest. 98:1063-1068 (1996)).
Prorenin or renin may have AII-independent actions to increase fibrotic disease. Prorenin overexpressing rats were found to be normotensive but to develop severe glomerulosclerosis (Veniant et al., J. Clin. Invest. 98:1996-1970 (1996)). Human recombinant renin added to human mesangial cells induces marked upregulation of production of plasminogen activator inhibitors (e.g. PAI-1) which block the generation of plasmin, a fibrinolytic enzyme important in the dissolution of clots after wounding-PAI-1 is increased in response to added TGF.beta. (Tomooka et al., Kidney Int. 42:1462-1469 (1992)), which is independent of AII and acts through a renin receptor on mesangial cells, independent of the enzymatic site used to convert angiotensin to angiotensinogen (Nguyen et al., Kidney Int. 50:1897-1903 (1996)). It has been suggested that TGF.beta. enhances renin release (Antonipillai et al., Am. J. Physiol. 265:F537-F541 (1993) and Ray et al., Contrib. Nephrol. 118:238-248 (1996)).
Thus, the interactions of RAS and TGF.beta. production form a complex system which impacts fibrotic ECM accumulation and the incidence of fibrotic disease. Various RAS components such as aldosterone, prorenin and renin may be connected with TGF.beta. production and fibrotic ECM accumulation. Any successful therapeutic regime must take into account these complex relationships to optimize inhibition of TGF.beta. to prevent and/or reduce ECM accumulation.
In fibrotic diseases overproduction of TGF.beta. results in accumulation of excess extracellular matrix which leads to tissue fibrosis and eventually organ failure. Accumulation of mesangial matrix is a histological indication of progressive glomerular diseases that lead to glomerulosclerosis and end-stage kidney disease (Klahr et al., N. Engl. J. Med. 318:1657-1666 (1988); Kashgarian and Sterzel, Kidney Int. 41:524-529 (1992)). Rats injected with antithymocyte serum are an accepted model of human glomerulonephritis and this model has demonstrated that overproduction of glomerular TGF.beta. can underlie the development of glomerulosclerosis (Okuda et al., J. Clin. Invest. 86:453-462 (1990); Border et al., Nature (Lond.) 346:371-374 (1990); Kagami et al., Lab. Invest. 69:68-76 (1993); and Isaka et al., J. Clin. Invest. 92:2597-2602 (1993)). Using cultured rat mesangial cells where the effects of Angiotensin II on glomerular pressure are not a factor, Angiotensin II has been shown to induce TGF.beta. production and secretion by mesangial cells, and this in turn has been shown to stimulate extracellular matrix production and deposition (Kagami et al., J. Clin. Invest. 93:2431-2437 (1994)). Increases in PAI-1 levels result in decreased degradation of extracellular matrix (Baricos et al., Kidney Int. 47:1039-1047 (1995)). Increases in TGF.beta. result in increased PAI-1 levels (Tomooka et al., Kidney Int. 42:1462-1469 (1992)). It has been demonstrated that decreasing TGF.beta. overexpression in a rat model of glomerulonephritis by in vivo injection of neutralizing antibodies to TGF.beta., reduces TGF.beta. overexpression (Border et al., Nature 346:371-374 (1990)), and reduces PAI-1 deposition into the pathological matrix (Tamooka et al., Kidney Int. 42:1462-1469 (1992)). Therefore, decreases in TGF.beta. levels should result in decreased PAI-1 levels and increased degradation of extracellular matrix to ameliorate organ impairment and fibrotic disease.
It is thus the aim of therapeutic strategies for fibrotic diseases to halt the overproduction of TGF.beta. and thus reduce the excess accumulation of extracellular matrix in tissues before organ failure occurs. There is a need for improved therapies that take into account the multiple pathways that stimulate TGF.beta. production.